Labelled sulfated amylopectins and method of determining abnormal gastrointestinal mucosa

ABSTRACT

Described herein are dye and radionuclide labelled anionic polysaccharide compositions, useful as diagnostic aids in the qualitative and quantitative detection in mammals of abnormal mucosa, particularly of the gastrointestinal variety, a process for their preparation and a method for their use. Preferred embodiments are the compositions of: sodium amylosulfate combined with technetium-99m and sodium amylosulfate combined with methylene blue.

United States Patent [191 [1 11 3,852,413 Cammarata Dec. 3, 1974LABELLED SULFATED AMYLOPECTINS [56] References Cited AND METHOD OFDETERMINING UNITED STATES PATENTS ABNORMAL GASTROINTESTINAL 3,637,6571/1972 Morii .1 260/2095 MUCOSA Inventor: Peter S. Cammarata, Skokie,111.

Assignee: G. D. Searle & Co., Chicago, 111.

Filed: July 3, 1972 Appl. No.: 268,641

Related US. Application Data Continuation-impart of Ser. No. 52,707,July 6, 1970, abandoned.

Foreign Application Priority Data July 2, 1971 Germany 2133005 US. Cl424/1, 250/303, 260/209.5,

424/7, 424/9 Int. Cl A6lk 27/04 Field of Search 424/1, 7, 9, 180;

250/106 T, 71.5 S; 260/2095; 23/203 B OTHER PUBLICATIONS Nuclear ScienceAbstracts, Vol. 24, No. 1, Jan. 15, 1970, page 87, item 767.

Primary Examiner-Benjamin R. Padgett Attorney, Agent, or FirmElliot N.Schubert [57] ABSTRACT Described herein are dye and radionuclidelabelled anionic polysaccharide compositions, useful as diagnostic aidsin the qualitative and quantitative detection in mammals of abnormalmucosa, particularly of the gastrointestinal variety, a process fortheir preparation and a method for their use. Preferred embodiments arethe compositions of: sodium amylosulfate combined with technetium99m andsodium amylosulfate combined with methylene blue.

i6 Claims, No Drawings LABELLED SULFATED AMYLOPECTINS AND METHOD OFDETERMINING ABNORMAL GASTROINTESTINAL MUCOSA This application forLetters Patent is a continuationin-part of Applicants prior copendingapplication, Ser. No. 52,707 filed July 6, 1970, now abandoned.

The detection and accurate measurement of abnormal mucosa (i.e. mucousmembrane), in mammals, especially humans, can be an invaluable aid tothe medical practitioner in the diagnosis of a great number of disordersassociated with the presence of such abnormal mucosa. Illustrative ofsuch disorders are gastric and duodenal ulcers, carcinoma, benignlesions comprehending those with diffuse mucosal changes frequentlyinterpreted as gastritis, oesophagitis, pre-ulcer, ulcerated colitis andsimilar pathological conditions. In the past, the most common methodemployed for the diagnosis of ulcers and stomach carcinoma was bariumradiology including the familiar barium meal followed by X-rayexamination. Unfortunately, the barium method suffers from severaldrawbacks including the well known dangers of X-ray examination, andinaccuracy and inconvenience in usage. For example, the bar ium methodis not susceptible of visualizing ulcers unless gross changes haveoccured in the mucosa and the ulcerated area is susceptible of planarvisualization. Consequently, it is difficult to detect gastric ulcersbecause it is frequently impossible to assess the dimensional aspects ofthe depth and angles of the abnormal area upon barium radiologicalexamination. Conversely, in certain subjects it has been found thatbarium radiology has shown abnormally deep rugal folds, particularly onthe greater curvature of the body of the stomach, supposedly indicativeof gastric ulcers, whereas the presence of ulceration could beconfidently excluded from the areas suspected upon appropriatefibroscopic examination, particularly utilizing as a diagnostic aid thelabelled compositions of this invention. In the case of X-rayexamination of the type employed in the barium meal method, i.e. wherethe radiological source of energy is outside the subject, incontradistinction to the source being within the subject as hereafterdescribed, there is the ever present danger of excess radiation whichcan be harmful to both the radiological technician and the subject,being particularly acute in the case of pregnant patients.

The object of this invention is to provide a safe, simplified, highlyaccurate, and convenient method for the detection in mammals of abnormalmucosa, particularly of the gastrointestinal variety, as well as alabelled composition suitable for use in this method. According to thismethod, a labelled polysaccharide composition, which binds strongly anddifferentially to mucosa, is administered to the mammal and timelysubsequent examination of the mammal by a suitable visualizing techniqueprovides definitive detection and quantification of abnormal mucosapresent in the tested area.

Polysaccharides suitable for the formulation of the compositions of thisinvention are anionic polysaccharides having a high charge density,gonveniently of the order of 0.97 X to 0.50 X 10 Z KA) (negative chargeper Angstrom") which characteristically bind differentially to mucosaand include sulfated and phosv phorylated polysaccharides having amolecular weight of at least 1,000,000. Among such sulfatedpolysaccharides are sulfated starch, sulfated mequite gum, sulfatedpsyllium seed, sulfated amylopectin, sulfated cellulose, sulfatedamylose and sulfated hyaluronic acid. Among the sulfated polysaccharideshaving specifically desirable application in this invention are thosecontaining one to two sulfate groups per monosaccharide unit, forexample per glucose unit in the case of starch, starch fractions, andparticularly corn and potato amylopectin. It has been observed thatpolysaccharides lacking a high charge density lack the ability of thepolysaccharide component of the instant compositions to differentiallyand strongly bind to mucosa, particularly without causing deleteriousside effects to the subject, for example degraded carrageenin.Particularly preferred sulfated polysaccharides for use in thisinvention are the alkali metal, and especially sodium, salts of sulfatedpotato starch amylopectin, said alkali metal salts possessing aboutl-1.8 sulfate groups per glucose unit and characterized also by amolecular weight of about 1-30 X 10 (The expression molecular weight asused in this application refers to weight average molecular weight.) Avery preferred polysaccharide is the sodium salt of sulfated potatostarch amylopectin possessing substantially 1.6 sulfate groups perglucose unit and characterized by a molecular weight of about 6.3 X 10which material is known by the non-proprietary name sodium amylosulfate.The preparation of the aforementioned alkali metal salts of sulfatedpotato starch amylopectin and in particular sodium amylosulfate, aredescribed in US. Pat. No. 3,271,388, issued Sept. 6, 1966. The lattersulfated polysaccharides are preferred components of the labelledcompositions of this invention because of their superior bindingproperties which promote not only their uniform binding with thelabelling agent, but also their differential adhesion to mucosa, whichmost importantly enables the resultant labelled compositions of thisinvention to be characterized by this same advantageous binding propertypermitting optimal physiological and diagnostic results.Physiologically, for example, the labelled composition of this inventionis not only safe, in view of its low toxicity and negligible absorptionfrom the gastrointestinal tract, but simultaneously provides therapeutictreatment for the abnormal area as well as amazingly accuratevisualization of the abnormality.

The choice of an appropriate labelling agent for use in the instantcomposition is dependent upon numerous factors which affect thediagnostic effectiveness and physiological suitability of the labellingagent and the ultimate composition. Moreover, each of these criticalfactors must be coordinated with the selection of polysaccharide andvisualizing instrument which is to be employed. Consequently,considering the magnitude and complexity of choices available, the safe,simplified, highly accurate and convenient labelled composition providedby this invention is most unexpected against the background of knownmaterials which have been employed for such purposes previously.Throughout this application the expression diagnostically effective" asapplied to the labelling agent of the instant composition shall refer tothat combination of properties, discussed with particularity hereinafterwith respect to both the dye and the radionuclide labelling agents,which enable the agent, as well as the composition in which it isincorporated, to permit definitive detection and quantification ofmucosal abnormalities by facilitating a satisfactory visualization ofthe abnormality by the imaging instrument employed. The expressionphysiologically suitable as applied to the labelling agent of theinstant composition refers to those properties discussed withparticularity hereinafter with respect to both the dye and radionuclidelabelling agents, which enable the agent, as well as the composition inwhich it is incorporated to produce a diagnostically effective responsewhile assuring patient and technician safety. It has been found that inview of their respective properties, certain radionuclides and certaindyes can meet the stringent requirements of diagnostically effective andphysiologically suitable and consequently constitute the appropriatelabelling agents of the instant compositions.

Specifically, in the selection of an appropriate dye for use in thisinvention from the standpoint of diagnostic effectiveness, one mustascertain that they exhibit strong light absorption and a capacity toprovide the desired contrast. Suitably such properties are present inmaterials having at least 1/10 the light absorption as does methyleneblue. As a measure of its absorption, it is noted that methylene bluehas a molecular extinction coefficient of 80,000 at 658 millimicrons inwater. In assessing physiological suitability, appropriate dyes arethose which have been found to substantially lack toxicity in mammals atdiagnostic doses, i.e. doses sufficient to permit visualization of theabnormality upon endoscopic examination, and which chemically combinewith the selected polysaccharide to form a uniformly stainedcomposition. Such dyes include basic aniline dyes, including azins(e.g., phenosafranine, methylene violet, diethylphenosafranine, etc.),pyronins (e.g., acridine red 38, pyronin B, etc.), oxazins (e.g.,brilliant cresyl blue, cresyl fast violet, nile blue lsulfatel), basicazo dyes (e.g., Janus green, indazole blue, Bismark brown, etc.),triphenyland diphenylnaphthylmethanes (e.g., malachite green,pararosaniline chloride, pararonsaniline acetate, methyl violet, ethylgreen, Victoria blue, etc.), phthalocyanines (e.g., alcian blue, luxolfast blue, etc.), and preferably the thiazins (e.g., thionin, azure A, Band C, methylene green, toluidine blue and especially methylene blue).Other polysaccharide staining dyes can also be used includingfluorochrome stains such as the fluoresceins, fat stains such as sudanblack B, the tetrazoles (e.g., blue tetrazolium), and food dyes such asF. D. and C. No. 1 (food and drug color No. 1). As indicated, thethiazins are the preferred dyes for the dye labelling component of theinstant diagnostic compositions, with methylene blue being mostpreferred, in view of their suitable extinction coefficient, which isboth strong as well as providing the desired contrast, and theirsubstantial lack of toxicity. A very preferred embodiment of thisinvention comprises the labelled composition containing preferably nogreater than 2 percent, but conveniently within the range of about 0.01to 5.0 percent, by weight of methylene blue and preferably no less than90 percent by weight of sodium amylosulfate. Said very preferredembodiment is especially advantageous as a diagnostic reagent incomparison with methylene blue used alone because of the formerssuperior binding qualities. That is, the intensity of color in thestained area is greater and persists longer than methylene blue whenemployed as a stain apart from the instant composition.

The choice of an appropriate radionuclide for use as an instantlabelling agent is likewise dependent upon its ability to bediagnostically effective and physiologically suitable. With respect to aradionuclide, the term physiologically suitable comprehends theproperties of: (1) short half life, (2) essentially pure gamma emissionand no substantial emission of beta-particles, and

' (3) chemical compatibility with the selected polysaccharide such thatnegligible absorption of the resultant composition occurs from thegastrointestinal tract. The

importance of a short half life, that is one no greater than 60 days andmore preferably of considerably low magnitude such as 2-24 hours, stemsfrom the fact that the radiation dose to the patient should be anintense burst of high energy in order to maximize effectiveness andsafety, since high energy radiation is better tolerated by mammals thanlow energy radiation. The use of an essentially pure gamma emittorprovides an additional safety factor because it has been observed thatradiation damage is frequently the result of the absorption of betaemissions, rather than gamma emissions. Generally gamma energy of 003-2Mev is desirable for an optimal and safe result. Among thosecharacteristics maximizing the diagnostic effectiveness of aradionuclide labelling agent is its chemical ability to bindirreversibly to the polysaccharide carrier. Such property is essentialto an effective diagnostic aid since less than essentially quantitativeand irreversible combination would provide: (1) an aggregate of label inthe composition which would not permit accurate visualization of theabnormality, or (2) loosely bound or unbound radionuclide or nuclidelabelled composition which would result in the loss of the materialthroughout the body of the mammal being studied and, perhaps,necessitate the administration of an unusually high dosage of oflabelled composition in order to achieve any useful visual result. Forobvious reasons of safety an unusually high radiation dosage is to beavoided. A significant factor which must be considered in the selectionof a diagnostically effective radionuclide for use in the instantlabelled composition is its ability to be visualized on existinginstrumentation. Many types of radiation imaging equipment providevarying tolerances of isotope energy, most commonly in the range of20-2000 Kev, though this wide range is not necessarily available on anyone instrument currently marketed. Among possible radionuclides for usein this invention are iodine-131, iodine-125, iodine-123, indium-l 1 l,indium- 113m, iodine-132, indium-114m, ytterbium-l69, gallium-67,dysprosium-l57, mercury-203, mercury-197, gold-198, bismuth-204,chromium-51 and most preferred, in view of its short half life, ideallack of beta emission and ability to be imaged on both rectilinearscanners and scintillation cameras, is technetium-99m. Additionalradionuclides, which because of unusually long half life, failure to beadequately imaged on present detectors, etc. are less desirable, thoughnevertheless of potential usefulness as advances in radiation imaginginstrumentation may provide better energy resolution enabling use oflower dosages of radionuclides, include selenium-75, gallium-68,cobalt-57, samarium- 153, and lutetium-177.

The dye labelled compositions can be manufactured by mixing the dye withthe selected polysaccharide in a suitable medium followed, if desired,by dialysis of the resulting product to enhance its purity.Alternatively other conventional methods of staining carbohydratematerials may be employed.

The radionuclide labelled compositions of this invention can be preparedby methods analagous to those used for the labelling of proteins, suchas albumin, and

fats such as triolein, as well as other radiolabelled carbohydratematerials known in the art. As an illustration of such a procedure thepreferred polysaccharide material of this invention, sodiumamylosulfate, was combined with l-sodium iodide in a suitable solventsuch as acetone, in the optional presence of an oxidizing agent, such as(N-chloro-p-toluenesulfonamido) sodium. After stirring and cooling, theresultant composition is optionally dialyzed against water to providegreater purity of the l-sodium amylosulfate composition product thereof.The technetium-99m labelled compositions of this invention are mostconveniently prepared by utilization of a reducing agent such as sodiumborohydride, ascorbic acid, a source of stannous ion, illustrativelystannous chloride, or most preferably a source of ferrous ion, such asferric chloride and ascorbic acid or ferrous chloride. The mostpreferred procedure'entails the addition of a few drops of hydrochloricacid to a saline solution of '"Tc-sodium pertechnetate, followed by theaddition of a reducing agent such as ferric chloride coupled withascorbic acid. The mixture is thereafter permitted to stand for severalminutes, after which time upon adjustment of pH, the desired sulfatedpolysaccharide, such as so dium amylosulfate, is added to the reactionmixture, and the pH suitably adjusted to provide the desiredtechnetium-99m labelled sodium amylosulfate. Any unbound technetium-99mmay be removed by dialysis or by passing a solution of the productthrough an ion exchange or gel filtration column.

As indicated above, the selection of radionuclide labelling agent forincorporation in the composition of this invention is dependent upon thevisualizing instrument which is to be used for the detection of abnormalmucosa. Described hereinafter are several types of radiation imagingdevices currently in use, each of which can be used with a least one ormore of the instant radionuclide labelled compositions. Specifically,the technetium-99m labelled compositions of this invention aredetectable on all of the instruments discussed hereinafter andconsequently these radionuclide labelled polysaccharides areparticularly preferred embodiments of this invention. Two basic types ofradiation imaging devices are rectilinear instruments or scanners, whosedetecting heads move back and forth over the scanning area; andscintillation cameras, which are characterized by their ability toproduce a unitary image of the object under study at any given moment.

With respect to the rectilinear scanner, the concentration ofradionuclide in the target organ is outlined by a corresponding patternof dots. Various black and white printout systems are often employed.However, systems in which the color of the printout changes with thecount rate may be of particular value when scanning areas of gradualchange in concentration. A typical rectilinear scanner isNuclear-Chicago Corporations PHO/DOT Isotope Scanner, whichautomatically produces a display of the location and concentration ofisotopelabelled compositions within selected organs or areas of thebody. Data is recorded on X-ray film by a photorecording system(photoscans) and is also printed on paper by a dot recording system(dotscans). The PHO/DOT has the ability to visualize isotopes within theenergy range of 202000 Kev.

The scintillation camera, commonly known as the Anger camera, isdescribed in its basic embodiment in US. Pat. No. 3,01 1,057. Suchcamera devices have the feature of viewing at least a substantialportion of the object under study at any one time, thus significantlyreducing examination time in comparison to rectilinear scanners. Atypical scintillation camera, Nuclear- Chicago Corporations PHD/GAMMAScintillation Camera, includes an image or detector head containing asodium iodide thallium activated scintillation crystal and a matrix ofphotomultiplier tubes; interchangeable collimators (multiaperture orpinhole); and image data computation, display and control modules. Thedistribution of radionuclides is often imaged on Polaroid film(scintiphotos). The PHO/GAMMA instrument has the ability to visualizeisotopes within the energy range of 50-680 Kev.

Very recent improvements of the scintillation camera are those havingtomographic imaging capabilities. These involve various additions to thebasic Anger camera system such as those described in Canadian Pat. Nos.872386 and 872387, in German Offenlegungsschrift No. 2011164 and in anarticle by G. Muehllehner, A Tomographic Scintillation Camera, Physicsin Medicine and Biology, Vol. 16., No. 1, pp. 87-96 (1971).Nuclear-Chicago Corporations PHOI- GAMMA Tomocamera Accessory Systemcomprises the conventional PHD/GAMMA Scintillation Camera, a rotatingslanted hole collimator assembly, a floatingtop radiographic table andan associated operating control module, and has the ability to imagedata from four distinct parallel enatomical planes in a single display.

The instant compositions can be combined with pharmaceuticallyacceptable carriers suitable for the administration of such a diagnosticaid. Obviously, the chemical properties of the particular labellingagent chosen for use in the composition will, in practice, limit thetype of carriers with which it can be combined. For example, the instanttechnetium-99m composition, because of the short half life oftechnetium-99m, can most conveniently be used as a liquid of relativelyfew ingredients, since compounding time for complicated formulationswould reduce the usefulness and practicality of the end product. Theterm pharmaceutically acceptable carrier as used herein in reference tothe compositions of this invention means a solid or a liquid composed ofa single substance or a number of substances which may be solids,liquids or a combination of solids and liquids each of which is lesstoxic than an equal weight of the active ingredient present in thecomposition when measured in the same mammalian host using the samemethod and conditions of administration. The concentration of the activeingredient in the composition is not critical, but for economy ofpreparation, when a dye is the labelling component, the concentrationshould be at least 0.5 percent by weight and preferably lpercent. Anillustration of a suitable radionuclide composition is that containing0.5 mg to 10 mg. of sodium amylosulfate per kilogram weight of themammalian patient species (1.7mg./kg. to 5mg./kg. being the morepreferred range) and 0.1 to 10 mCi milliCuries of a radionuclide such astechnetium-99m (more preferably in the amount of 2 milliCuries). Thesecompositions can be administered either orally or parenterally. For oraladministration, tablets, lozenges, capsules, dragees, pills and powdersare suitable, while aqueous and non-aqueous solutions or suspensions areappropriate for both oral (e.g. in the case of radionuclide labellingagents via a stomach tube) and parenteral (e.g. rectal) administration.Acceptable pharmaceutical carriers are exemplified by chewable tablets,sugars such as lactose or sucrose, starches such as corn starch orpotato starch, cellulose derivatives such as sodium carboxymethylcellulose, ethyl cellulose, methyl cellulose or cellulose acetatephthalate, talc, calcium phosphates such as dicalcium phosphate ortricalcium phosphate, sodium sulfate, calcium sulfate, polyvinylalcohol, acacia, stearic acid, alkaline earth metal stearates such asmagnesium stearate, vegetable oils such as peanut oil, cottonseed oil,sesame oil, olive oil, corn oil, and oil of theobroma, water, agar,alginic acid, benzyl alcohol, isotonic saline and phosphate buffersolution as well as other non-toxic compatible substances used inpharmaceutical formulations. Some typical formulations as well as themethod of preparation thereof, are presented below.

synth entrapped black cherry flavor, the mixture was blended for 5minutes and a sample was taken for assaying. Thereafter it wascompressed into tablets of the appropriate size.

The method of this invention for the detecting in mammals of abnormalmucosa, particularly of the gastrointestinal variety, comprises theadministration of a diagnostic dose of the instant novel diagnosticcomposition. The term diagnostic dose is defined as the amount of activeingredient that will adequately adhere to the mucosa and effectlabelling thereof sufficient to permit detection upon examination,endoscopically in the case of dye labelling agents and by radiationimaging devices in the instance of radionuclide labelling agents. Thisdose will vary with the particular labelling agent chosen, the type andlocation of the particular disorder being diagnosed, the route ofadministration,

Formulation l Liquid Composition Containing 2% Methylene Blue:

Sodium Amylosulfate Amount ingredient (Conc. 250 mg/5 ml.)

(Conc. 500 mg/5 ml.)

Distilled Water 5 ml.

5 int.

To the composition of methylene blue with sodium amylosulfate was addedslowly, with stirring, 100 ml. of distilled water. To this slurry wasadded, with stirring, sufficient distilled water to bring the mixture tothe volume of 1 liter and stirring was continued until solution wascomplete. The solution was then poured into amber bottles of the desiredquantity, for example, 5 milliliter portions, sampled and assayed.

Formulation ll Chewable Tablet Composition Containing 2% Methylene Blue:Sodium Amylosulfate Amount Ingredient Per Tablet Per Batch ofComposition of methylene 500 mg. 500 g. blue with sodium amylosulfate(in a ratio of pre ferably 2zl00, respectively) Dextrose USP, anhydrous1.66 mg. 1660 g. Polyvinylpyrrolidone 67.4 mg. 67.4 g, Hydrogenatedcottonseed oil 22.6 mg, 22.6 g. Flavor (e.g. Florasynth en- 1.76 mg.1.76 g.

trapped black cherry flavor) drogenated cottonseed oil and 1.76 grams ofFlorathe particular polysaccharide used, and the subjects physicalcharacteristics, e.g. body weight and physiological state such ashypersecretory. A typical diagnostic dosage for a subject of about 22 toabout 136 kg. body weight for the composition of sodium amylosulfatewith up to 2 percent methylene blue is to 500 milligrams, administeredabout /4 hour prior to endoscopic examination for gastric ulcer. For asubject of identical weight and disorder, 2 milliCuries of techetium-99mcombined with 250 mg. of sodium amylosulfate, administered l2 hoursprior to examination with a radiation imaging device, would be a typicaldiagnostic dosage.

Application of the instant method to the diagnosis of abnormal gastricmucosa, particularly ulcers, has been found to be especially effectivewhen the preferred dye labelled composition of this invention, i.e.sodium amylosulfate with no greater than 2 percent by weight methyleneblue, is administered to the patient 15 minutes prior to endoscopicexamination employing a gastroscope (i.e. a hollow tubular instrumentdesigned to pass into the stomach by way of the mouth and esophagus andfitted with optical and lighting equipment that permits visualinspection of the stomach) to which a camera suitable for colorphotography has been affixed. As will be apparent to those skilled inendoscopic identification, the light source for the gastroscope may bevisible or ultraviolet lighting dependent upon the particular dyeemployed in the composition. In a very preferred embodiment of thisinvention, methylene blue, which may be viewed by visible light, servesas the dye.

In one clinical illustration of this very preferred method anddiagnostic aid of this invention, a human subject, previously known topossess an ulcer as diagnosed by barium radiology, was administered 5ml. of

an aqueous solution containing 250 mg. of the composition comprising 2percent methylene blue with sodium amylosulfate. Fifteen minutes later,gastroscopic examination of the patient accompanied by color photographyof the area in question revealed the ulcer. The visualization of theulcer, both to the naked eye and in the color photographs, was marked bysharpness of' color, namely bright blue, at the edges of the ulcercrater, while the crater was differentially deprived of such color.Moreover, the shape and character of the ulcer as well as an indicationof depth was apparent upon inspection. Thus, in contract and preferenceto X-ray pictures produced via the barium meal method, wherein thecrater itself is often poorly defined or distorted, the shapeand'character of the ulcer or other abnormality in the mucosa is easilydetected and quantitied. Hence the instant diagnostic composition andmethod are particularly useful in detecting aberations of gastric mucosasince the characteristics of the stomach (its asymmetry, flabbiness andvariability in shape and motility) make it difficult to examine byradiological techniques employing a source of radioenergy ouside thesubject, such as the barium meal method. Most effective and desirableresults are obtained when the radioenergy source is placed within thesubject, as with the instant compositions wherein the labelling agentisa radionuclide, most preferably technetium-99m, as i1- lustrated inExamples 3 to 11 described hereinafter.

The following examples are given for the purpose of illustrating thepreparation of the instant diagnostic composition according to thepresent invention. It will be understood that the invention is not to beconstrued as limited in spirit or in scope by the details containedtherein, as many modifications in materials and methods will be apparentfrom this disclosure to those skilled in the art. In the followingexamples, temperatures are given in degrees Centigrade (C.)

EXAMPLE 1 A 1 percent aqueous solution of sodium amylosulfate. (i.e.,the sodium salt of sulfated potato starch amylopectin, possessingsubstantially 1.6 sulfate groups per glucose unit, characterized bymolecular weight of about 6.3 X and having been prepared according tothe method described in Example 4 of U.S. Pat. No. 3,271,388) was placedinside a dialysis bag, and the bag was then placed inside a containerhaving a 0.5 percent solution of methylene blue (U.S.P.) in water. Theratio of methylene blue solid, in the outer compartment, to sodiumamylosulfate solid, in the inner compartment, was 2/ 100. The twosolutions were allowed to equilibrate for 24 hours, after which timepractically all the methylene blue had diffused into the dialysis bagcontaining the sodium amylosulfate solution. At the end of this time,the dialysis bag was removed from the exhausted methylene blue outersolution and placed in a large container of hydrochloric acid at pH 2,where it 1 was dialyzed for 24 hours. No detectable methylene .blue wasremoved by 24 hour dialysis against the acid solution, proving theirreversible binding of methylene blue to sodium amylosulfate under acidconditions analogous to that of gastric juice. At the end of the aciddialysis period, the contents of the dialysis bag were neutralized to apH of 7.4 with sodium hydroxide and lyophilized.

Alternatively, upon completion of the 24 hour equilibration describedabove, the dialysis bag containing the sodium amylosulfate-methyleneblue solution was removed from the exhausted methylene blue outersolution and was adjusted to a pH of 7.8 with sodium hydroxide and thesolution was thereafter lyophilized for 72 hours. The lyophilizedmaterial was first extracted with ethanol and then with redistilledacetone, followed by filtration. The resultant filtercake was dried invacuo for 48 hours to provide a composition, identical with thatprepared in the preceding paragraph containing 2 percent methylene bluewith about 93 percent sodium amylosulfate, which composition ischaracterized by ultraviolet absorption maxima at about 246, 285 and 575millimicrons. It will be observed that the methylene blue componentindividually displays ultraviolet absorption maxima at about 609 and 667millimicrons. Thus by comparison with the maxima described above for theprepared composition, this metachromatic be havior is evidence that thecomposition consists of a unique composition of matter distinguishablefrom its component, methylene blue.

EXAMPLE 2 A 0.1 percent solution of methylene blue in water was pumpedslowly, over a 48 hour period, at a rate of 0.1 ml./min., into a rapidlystirred 1.0 percent solution of the alkali metal salt of sulfated potatostarch amylopectin possessing 1-1.8 sulfate groups per glucose unit andbeing characterized also by a molecular weight of 1-30 X 10 in water.After the solutions were combined, the dyed salt solution was dialyzedagainst acid, then against water, and finally lyophilized and purifiedas described in the above example to provide a composition containing 2percent methylene blue and an alkali metal salt of sulfated potatostarch amylopectin possessing 11.8 sulfate groups per glucose unit andbeing characterized also by a molecular weight of 1-30 x 10 EXAMPLE 3 Toa suspension of 503 mg. of sodium amylosulfate in 10 ml. of acetone wasadded a solution of l mCi of carrier-free 'l-sodium iodide in 10 ml. ofacetone. To that mixture was then added 10 p1. of an aqueous solution ofChloramine-T, i.e. (N-chloro-ptoluenesulfonamido)sodium [concentration 2mg./ml.], and the resultant mixture was stirred at C. for about 2 hours,then allowed to cool to room temperature over a period of 1 hour. Themixture was diluted with approximately 50 ml. of water and then dialyzedagainst three 5.5 liter portions of water during a period of 4 days,using cellulose dialyzer tubing. The contents of the tubing were thenlyophilized, affording approximately 0.45 g. of l-sodium amylosulfatecomposition having a specific activity of 18.9 pCi/g.

A solution containing the l-sodium amylosulfate product described abovein 50 ml. of water was administered intragastrically to a 7.8 kg. femaledog (Beagle). After waiting 5 minutes, the stomach region was visualizedby means of a Nuclear-Chicago Corporation PHO/DOT Isotope Scanner. Thedot and photo scans demonstrated the binding effect of l-sodiumamylosulfate to the muscosa observed.

EXAMPLE 4 To a suspension of 503 mg. of sodium amylosulfate to 10 ml. ofacetone was added a solution of 1 mCi of carrier-free l-sodium iodide in10 ml. of acetone. That mixture was stirred at 60C. for aboutZ hours,then allowed to cool to room temperature over a period of 1 hour. Theresultant mixture was transferred to dialysis tubing with approximately2 ml. of acetone and 60 ml. of water and then dialyzed against 5.5liters of water for about 2 hours. The contents of the tubing werelyophilized. There were obtained approximately 409 mg. of l-sodiumamylosulfate composition having a specific activity of 18.0 ,uCi/g.

EXAMPLE 5 501.5 Mg. of sodium amylosulfate, 5.1 ml. of water containing1 mCi of carrier-free l-sodium iodide, and an aqueous solution of 4.4 Xmillimoles of Cloramine-T were combined and the required amount of waterneeded to bring the total amount of water to 50 ml. was added. Theresulting mixture, containing lsodium amylosulfate, was administeredintragastrically to a 7.8 kg. dog (Beagle) and the stomach region wasvisualized by means of a Nuclear-Chicago Corporation PRO/GAMMAScintillation Camera. Scintiphotos were taken over a period of 2V2hours. Binding to the gastrointestional mucosa was observed throughoutthe length of the visualization period.

EXAMPLE 6 A solution of 3.69 mCi of carrier-free '"Tc-sodiumpertechnetate in 2 ml. of saline (obtained by elution from a Mo- "Tcgenerator) was acidified with 3 drops of 2 N aqueous hydrochloric acidsolution. To that solution were added 10 mg. of ferric chloridehexahydrate, followed by 8 mg. of ascorbic acid. The acidity of theresulting solution was adjusted to approximately pH 5 by addition of 14drops of 1N aqueous sodium hydroxide solution. 500 Mg. of sodiumamylosulfate were then added and the mixture was stirred for about 5minutes. At the end of that time, the mixture was passed through a smallSephadex chromatographic column in order to remove free technetium. Thefirst 25 ml. of eluate were discarded and the next 50 ml. werecollected. The latter eluate contained '"Tc-sodium amylosulfate with anactivity, corrected to zero time, of 0.0426 mCi/ml. (total radioactivity2.1 mCi). 43 Ml. of that solution (radioactivity 1.8 mCi) wereadministered to a 7.8 kg. dog (Beagle) for gastrointestinalvisualization using a Nuclear-Chicago Corporation PHO/- GAMMAScintillation Camera. Scintiphotos were taken beginning at about 4minutes after administration and continuing for 3 /2 hours. Binding tothe gastrointestinal mucosa was observed throughout the length of thevisualization period.

EXAMPLE 7 A solution of 3.4 mCi of carrier-free Tc-sodium pertechnetatein 2 ml. of saline (obtained by elution from a "Mo-"Tc generator) wasstirred with 500 mg. of sodium amylosulfate in 50 ml. of water for 5minutes. The resultant mixture was poured into dialysis tubing anddialyzed against three 5.5 liter portions of water during a period ofabout 5% hours. There was thus obtained 25 ml. of a '"Tc-sodiumamylosulfate composition having a total radioactivity, corrected to zerotime, of about 0.12 mCi.

EXAMPLE 8 A solution of 3.4 mCi of carrier-free "Tc-sodium pertechnetatein 2 ml. of saline (obtained by elution from a Mo- Tc generator) wasstirred while adding 3 drops of 2 N aqueous hydrochloric acid solution.There were then added, with stirring, 10 mg. of ferric chloride,followed by 8.5 mg. of ascorbic acid. The acidity of the resultingsolution was adjusted to pH 4.5-5.5 with 1 N aqueous sodium hydroxidesolution. 503 Mg. of sodium amylosulfate were added and stirring wascontinued for about 30 minutes. The resultant solution was transferredto dialysis tubing and dialyzed against three 4 liter portions of waterduring a period of about 5% hours. There were obtained 28 ml. of a'"Tc-sodium amylosulfate composition having a total radioactivity,corrected to zero time, of 1.24 mCi.

EXAMPLE 9 A solution of 3.86 mCi of carrier-free Tc-sodium pertechnetatein 2 ml. of saline (obtained by elution from a Mo- Tc generator) wasacidified with 3 drops of 2 N aqueous hydrochloric acid solution. Tothat solution were added 17.5 mg. of ferric chloride, followed by 16 mg.of ascorbic acid. The acidity of the resulting solution was adjusted topH 4.55.5 by the addition of approximately 14 drops of 1 N aqueoussodium hydroxide solution. 500 Mg. of sodium amylosulfate were thenadded and the mixture was stirred for about 1 hour. At the end of thattime, the mixture was passed through a small Sephadex column. The first20-30 ml. eluted from the column were discarded. The next 50 ml. whichwere eluted were collected. There were thus obtained 50 ml. of a"'Tc-sodium amylosulfate composition having a total radioactivity,corrected to zero time, of 3.36 mCi.

EXAMPLE 10 A solution of 3.86 mCi of carrier-free "Tc-sodiumpertechnetate in 2 ml. of saline (obtained by elution from a Mo- Tcgenerator) was acidified with 3 drops of 2 N aqueous hydrochloric acidsolution. To that solution were added 17.5 mg. of ferric chloride,followed by 16 mg. of ascorbic acid. The acidity of the resultingsolution was adjusted to pH 4.5-5.5 by the addition of approximately 14drops of 1 N aqueous sodium hydroxide solution. 500 Mg. of the sodiumsalt of sulfated potato starch amylopectin possessing 1.8 sulfate groupsper glucose unit and being characterized also by a molecular weight of12.5 X 10 were then added and the mixture was stirred for about 1 hour.At the end of that time, the mixture was passed through a small Sephadexcolumn. The first 25 ml. eluted from the column were discarded. The next50 ml. which were eluted were collected. There were thus obtained 50 ml.of a composition containing the sodium salt of sulfated potato starchamylopectin possessing 1.8 sulfate groups per glucose unit and beingcharacterized by a molecular weight of 12.5 X 10", labelled withtechnetium-99m.

EXAMPLE 11 A solution of 3.69 mCi of carrier-free 'lc-sodiumpertechnetate in 2 ml. of saline (obtained by elution from a "Mo' Tcgenerator) was acidified with 3 drops of 2 N aqueous hydrochloric acidsolution. To that solution were added 10 mg. of ferric chloride,followed by 8 mg. of ascorbic acid. The acidity of the resultingsolution was adjusted to approximately pH 5 by the addition of 14 dropsof 1 N aqueous sodium hydroxide solution. 500 Mg. of the alkali metalsalt of sulfated potato starch amylopectin possessing 1-1.8 sulfategroups per glucose unit and being characterized also by a molecularweight of 130 X were then added and the mixture was stirred for about 5minutes. At the end of that time, the mixture was passed through a smallSephadex column. The first ml. of eluate were discarded and the next 50ml. were collected. The latter eluate contained the aforementionedalkali metal salt of sulfated potato starch amylopectin, labelled withtechnetium-99m.

What is claimed is:

l. A composition comprising an alkali metal salt of sulfated potatostarch amylopectin, which is characterized by a molecular weight ofabout 1-30 X 10 and a sulfate content of about ll.8 sulfate groups perglucose unit, chemically combined with a diagnostically effective,physiologically suitable labelling agent.

2. The composition according to claim 1 wherein the alkali metal salt isthe sodium salt possessing about 1.6 sulfate groups per glucose unit andcharacterized also by a molecular weight of about 6.3 X 10.

3. A composition according to claim 1 wherein the alkali metal salt isthe sodium salt possessing about 1.8 sulfate groups per glucose unit andcharacterized also by a molecular weight of about 12.5 X 10".

4. The composition according to claim 1 wherein the diagnosticallyeffective, physiologically suitable labelling agent is a thiazin dye.

5. The composition according to claim 1 wherein the diagnosticallyeffective, physiologically suitable labelling agent is methylene blue.

6. A composition according to claim 1 comprising an alkali metal salt ofsulfated potato starch amylopectin, which is characterized by amolecular weight of 1-30 X 10 and a sulfate content of l-l .8 sulfategroups per glucose unit chemically combined with 0.01 to 5 percent byweight of methylene blue.

7. A composition according to claim 1 wherein the diagnosticallyeffective, physiologically suitable labelling agent is a radionuclide.

8. A composition according to claim 1 wherein the diagnosticallyeffective, physiologically suitable labelling agent is iodine-125.

9. The composition according to claim 1 wherein the diagnosticallyeffective, physiologically suitable labelling agent is iodine-131.

10. The composition according to claim 1 wherein the diagnosticallyeffective, physiologically suitable labelling agent is technetium99m.

11. A composition comprising the sodium salt of sulfated potato starchamylopectin, which is characterized by a molecular weight of about 6.3 X10 and a sulfate content of about 1.6 sulfate groups per glucose unit,chemically combined with technetium-99m.

12. A method of detecting abnormal gastrointestinal mucosa whichcomprises administering to a mammal a diagnostic dose of a compositioncomprising a sulfated polysaccharide, which differentially binds to thegastrointestinal mucosa, and which is characterized by a molecularweight of at least one million and a sulfate content of substantiallyl-2 sulfate groups per monosaccharide unit, chemically combined with adiagnostically effective, physiologically suitable labelling agent,followed by examination of the mucosa in order to determine thoseportions containing bound labelled sulfated polysaccharide.

13. The method according to claim 12 wherein the polysaccharide is analkali metal salt of sulfated potato starch amylopectin, said saltpossessing about ll.8 sulfate groups per glucose unit and characterizedalso by a molecular weight of about 1-30 X 10 14. The method accordingto claim 12 wherein the diagnostically effective, physiologicallysuitable labelling agent is methylene blue, which is visualized uponendoscopic examination of the mucosa in order to determine thoseportions containing bound labelled sulfated polysaccharide.

15. The method according to claim 12 wherein the diagnosticallyeffective, physiologically suitable labelling agent is technetium-99mand the examination is conducted with a radiation imaging device inorder to determine those portions containing bound labelled sulfatedpolysaccharide.

16. The method of detecting abnormal gastrointestinal mucosa in mammalswhich comprises administering to the mammal a diagnostic dose of acomposition comprising the sodium salt of a sulfated potato starchamylopectin possessing 1.8 sulfate groups per glucose unit andcharacterized also by a molecular weight of 6.3 X 10', chemicallycombined with the diagnostically effective, physiologically suitablelabelling agent, technetium-99m, followed by examination with aradiation imaging device in order to determine those portions containingbound labelled sulfated polysaccharide.

1. A COMPOSITION COMPRISING AN ALKALI METAL SALT OF SULFATE POTATOSTARCH AMYLOPECTIN, WHICH IS CHARACTERIZED BY A MOLECULAR WEIGHT OF AOUT1-30X10**7 AND A SULFATE CONTENT OF ABOUT 1-1.8 SULFATE GROUPS PERGLUCOSE UNIT, CHEMICALLY COMBINED WITH A DIAGNOSTICALLY EFFECTIVEPHYSIOLOGICALLY SUITABLE LABELLING AGENT.
 2. The composition accordingto claim 1 wherein the alkali metal salt is the sodium salt possessingabout 1.6 sulfate groups per glucose unit and characterized also by amolecular weight of about 6.3 X
 107. 3. A composition according to claim1 wherein the alkali metal salt is the sodium salt possessing about 1.8sulfate groups per glucose unit and characterized also by a molecularweight of about 12.5 X
 107. 4. The composition according to claim 1wherein the diagnostically effective, physiologically suitable labellingagent is a thiazin dye.
 5. The composition according to claim 1 whereinthe diagnostically effective, physiologically suitable labelling agentis methylene blue.
 6. A composition according to claim 1 comprising analkali metal salt of sulfated potato starch amylopectin, which ischaracterized by a molecular weight of 1-30 X 107 and a sulfate contentof 1-1.8 sulfate groups per glucose unit chemically combined with 0.01to 5 percent by weight of methylene blue.
 7. A composition according toclaim 1 wherein the diagnostically effective, physiologically suitablelabelling agent is a radionuclide.
 8. A composition according to claim 1wherein the diagnostically effective, physiologically suitable labellingagent is iodine-125.
 9. The composition according to claim 1 wherein thediagnostically effective, physiologically suitable labelling agent isiodine-131.
 10. The composition according to claim 1 wherein thediagnostically effective, physiologically suitable labelling agent istechnetium-99m.
 11. A composition comprising the sodium salt of sulfatedpotato starch amylopectin, which is characterized by a molecular weightof about 6.3 X 107 and a sulfate content of about 1.6 sulfate groups perglucose unit, chemically combined with technetium-99m.
 12. A method ofdetecting abnormal gastrointestinal mucosa which comprises administeringto a mammal a diagnostic dose of a composition comprising a sulfatedpolysaccharide, which differentially binds to the gastrointestinalmucosa, and which is characterized by a molecular weight of at least onemillion and a sulfate content of substantially 1-2 sulfate groups permonosaccharide unit, chemically combined with a diagnosticallyeffective, physiologically suitable labelling agent, followed byexamination of the mucosa in order to determine those portionscontaining bound labelled sulfated polysaccharide.
 13. The methodaccording to claim 12 wherein the polysaccharide is an alkali metal saltof sulfated potato starch amylopectin, said salt possessing about 1-1.8sulfate groups per glucose unit and characterized also by a molecularweight of about 1-30 X
 107. 14. The method according to claim 12 whereinthe diagnostically effective, physiologically suitable labelling agentis methylene blue, which is visualized upon endoscopic examination ofthe mucosa in order to determine those portions containing boundlabelled sulfated polysaccharide.
 15. The method according to claim 12wherein the diagnostically effective, physiologically suitable labellingagent is technetium-99m and the examination is conducted with aradiation imaging device in order to determine those portions containingbound labelled sulfated polysaccharide.
 16. The method of detectingabnormal gastrointestinal mucosa in mammals which comprisesadministering to the mammal a diagnostic dose of a compositioncomprising the sodium salt of a sulfated potato starch amylopectinpossessing 1.8 sulfate groups per Glucose unit and characterized also bya molecular weight of 6.3 X 107, chemically combined with thediagnostically effective, physiologically suitable labelling agent,technetium-99m, followed by examination with a radiation imaging devicein order to determine those portions containing bound labelled sulfatedpolysaccharide.